Medium and Method for Culturing Mycobacterium Avium Subspecies Paratuberculosis

ABSTRACT

The present invention provides a growth medium and kit containing a nicotine analog and use of the same in a method for enhancing the growth of  Mycobacterium avium  subspecies  paratuberculosis  (MAP).

This application claims the benefit of priority from U.S. ProvisionalSer. No. 61/246,227 filed Sep. 28, 2009, which is incorporated herein inits entirety.

BACKGROUND OF THE INVENTION

The use of tobacco products has empirically observed, disparate effectsin inflammatory bowel disease (IBD) (Birrenbach & Bocker (2004) Inflamm.Bowel Dis. 10:848-859; Karban & Eliakim (2007) World J. Gastroenterol.13:2150-2152; Regueiro, et al. (2005) Inflamm. Bowel Dis. 11:42-47).Crohn's disease (CD) (Dalziel (1913) Br. Med. J. ii:1068-1070; Crohn, etal. (1932) J. Amer. Med. Assoc. 99:1323-1328) is exacerbated by the useof tobacco products (Regueiro, et al. (2005) supra; Cosnes, et al.(1999) Aliment Pharmacol. Ther. 13:1403-1411; Lewis, et al. (2007) J.Med. Genet. 44:689-694). In contrast, the clinical course of ulcerativecolitis (UC) is ameliorated by use of tobacco (Regueiro, et al. (2005)supra; Aldhous, et al. (2007) Am. J. Gastroenterol. 102:589-597;Beaugerie, et al. (2001) Am. J. Gastroenterol. 96:2113-2116), ornicotine (Pullan, et al. (1994) N. Engl. J. Med. 330:811-815; McGrath,et al. (2004) Cochrane Database Syst. Rev.:CD004722) one of tobacco's4000 constituent molecules (Dube & Green (1982) 36^(th) Tobacco ChemistsResearch Conference. Symposium on the Formation, Analysis andComposition of Tobacco Smoke, Raleigh N.C. pp. 42-102; Jenkins, et al.(2000) The Chemistry of Environmental Tobacco:Composition andMeasurement; Eisenberg (ed.) Boca Raton Fla.:CRC Press). Themechanism(s) involved although intensively investigated (Aldhous, et al.(2009) PLoS ONE 4:e6285; Nielsen, et al. (2009) PLoS ONE 4:e6210) is notunderstood (Karban & Eliakim (2007) supra), but is assumed to be due tothe most bioactive component of tobacco; nicotine (Karban & Eliakim(2007) supra; Aldhous, et al. (2009) supra). It is of note that in aprior study, involving a solitary MAP strain, pure nicotine inhibitedMAP growth in culture (Naser, et al. (2001) Am. J. Gastroenterol.96:3455-3457).

The etiology of CD and UC is (are) not known. Mycobacterium aviumsubspecies paratuberculosis (MAP), causes a chronic wasting diarrhealdisease in cattle called Johne's disease (Johne & Frothingham (1895)Dtsch. Zeitschr. Tiermed., Vergl. Pathol. 21:438-454), that is evocativeof CD. Humans are continually exposed to viable MAP (Mishina, et al.(1996) Proc. Natl. Acad. Sci. USA 93:9816-9820; Ellingson, et al. (2005)J. Food Prot. 68:966-972; Grant, et al. (2002) Appl. Environ. Microbiol.68:602-607; Ayele, et al. (2005) Appl. Environ. Microbiol.71:1210-1214). There is increasing concern that MAP may be zoonotic(Greenstein & Collins (2004) Lancet 364:396-397; Greenstein (2003)Lancet Infect. Dis. 3:507-514; Greenstein, et al. (2009) In: Fratamicoet al. (eds.) Sequelae and Long-Term Consequences of InfectiousDiseases. One ed. Washington, ASM Press. American Society forMicrobiology pp. 135-168). In contrast to leprosy, where M. leprae hasnever been grown in vitro (Stewart-Tull (1982) In: Ratledge & Stanford(eds.) The Biology of the Mycobacteria, Volume 1: Physiology,Identification, and Classification. One ed. New York, Academic Press.pp. 273-307), MAP has been cultured from humans with CD (Chiodini, etal. (1986) J. Clin. Microbiol. 24:357-363; Chiodini, et al. (1984) J.Clin. Microbiol. 20:966-971; Naser, et al. (2004) Lancet 364:1039-1044;Naser, et al. (2000) Am. J. Gastroenterol. 95:1094-1095; Bull, et al.(2003) J. Clin. Microbiol. 41:2915-2923) as well as patients with UC(aser, et al. (2004) supra).

There is an emerging explanation as to why this probable MAP zoonosishas not been appreciated. It is that multiple agents used in thetreatment of IBD are in fact anti-MAP antibiotics. They areconventionally called “anti-inflammatories” (Greenstein, et al. (2007)PLoS ONE 2:e516) and “immuno modulators” (Shin & Collins (2008)Antimicrob. Agents Chemother. 52:418-426; Greenstein, et al. (2009) Int.J. Infect. Dis. 13:e254-263; Greenstein, et al. (2007) PLoS ONE 2:e161;Greenstein, et al. (2008) PLoS ONE 3:e249). Analogous to the multipleclinical manifestations of leprosy (Greenstein, et al. (2009) supra;Ridley & Jopling (1962) Lepr. Rev. 33:119-128; Ridley & Jopling (1966)Int. J. Lepr. Other Mycobact. Dis. 34:255-273), it has been suggestedthat all of IBD may be caused MAP (Mishina, et al. (1996) supra;Greenstein, et al. (2009) supra; Naser, et al. (2004) supra).

In many cases, the detection of MAP in living tissue, food, and waterrequires first culturing the bacterium. Even though MAP is hardy, it isslow growing and fastidious, which means it is difficult to culture. Tofacilitate detection of MAP, improved culturing methods are needed. Thepresent invention addresses this need in the art by providing a culturemedium which enhances the growth of MAP thereby facilitating itsdetection in food, water, and other biological samples.

SUMMARY OF THE INVENTION

The present invention features a method for enhancing the growth of MAPby culturing MAP in the presence of a nicotine analog. In someembodiments, the nicotine analog has the structure

wherein R₁ is NH₂ or O and R₂ is absent, or a ribose or nucleotidegroup. In particular embodiments, the nicotine analog is nicotinic acid,nicotinamide, α-NAD, or β-NAD. In yet other embodiments, the MAP is froma biological sample.

The present invention also features a growth medium and kit containingat least one nicotine analog for use in enhancing the growth of MAP.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the cumulative Growth Index (cGI) of MAP strain Dominicin the presence of nicotine, nicotinic acid, α-nicotinamide adeninedinucleotide (NAD), β-NAD, and isoniazid, expressed as.

FIG. 2 depicts the depicts the cumulative Growth Index (cGI) of MAPstrain UCF-4 in the presence of nicotine, nicotinic acid, nicotinamide,α-NAD, β-NAD, and isoniazid as compared to control (C).

FIG. 3 shows the cumulative Growth Index (cGI) growth of M. tuberculosisin the presence of nicotine, nicotinic acid, nicotinamide, α-NAD, β-NAD,and isoniazid as compared to control (C).

DETAILED DESCRIPTION OF THE INVENTION

Tobacco use has been shown to exacerbate Crohn's disease and itscessation results in clinical improvement (Kane, et al. (2005) J. Clin.Gastroenterol. 39(1):32-5). Therefore, it was determined whethernicotine, or its structural analogs could modify MAP growth kinetics invitro. In this analysis, MAP was grown in the presence of varyingconcentrations of nicotine, isoniazid, nicotinic acid, nicotinamide,α-NAD, and β-NAD. The results of this analysis indicated that whilenicotine had no effect on the growth kinetics at the concentrationstested, nicotine structural analogs, nicotinic acid, nicotinamide,α-NAD, and β-NAD, profoundly effected the growth kinetics of multiplestrains of MAP including Dominic (FIG. 1) and UCF-4 (FIG. 2). Incontrast, nicotine and its structural analogs provided no enhancement inthe growth of M. tuberculosis complex (FIG. 3). In light of theseresults, nicotine analogs find use in the selective enhancement of MAPgrowth in vitro.

Thus, the present invention embraces a MAP culture medium, kit andmethod for enhancing the growth of MAP. According to the method of theinvention, MAP is cultured in the presence of a nicotine analog so thatgrowth of MAP is enhanced. For the purposes of the present invention,culturing of MAP refers to the growth of MAP bacterium in vitro in asolid, semi-solid or liquid medium. While exemplary media are providedherein, it is contemplated that any medium known in the art to supportthe growth of MAP can be supplemented with one or more nicotine analogsaccording to the present invention.

Culturing of MAP can be for various purposes including, but not limitedto, the identification of anti-MAP agents, the detection of MAP inbiological samples, the diagnosis of a MAP infection, as well as inresearch of morphological, physiological and molecular biologicalaspects of MAP. As such, MAP can be obtained from a variety of sources,including biological samples such as food, water, blood, a stool sample,cerebrospinal fluid or alternatively a biopsy sample, e.g., lesionedcentral nervous tissue or a biopsy obtained in endoscopy or a surgicallyressected specimen. In vitro culturing of MAP bacterium generallyinvolves placing the sample on an appropriate growth medium underconditions suitable for growth of MAP. By way of illustration, MAP canbe obtained by harvesting a tissue sample from a subject suspected ofhaving a MAP infection (e.g., a subject suspected of having Crohn'sdisease, inflammatory bowel disease, Multiple Sclerosis or Alzheimer'sDisease), washing the tissue sample with phosphate-buffered saline(e.g., 0.067 M, pH 6.8) and resuspending the sample in albumin. Thesample is then inoculated into MGIT media (Becton Dickinson, Palo Alto,Calif.) supplemented with a nicotine analog, and the medium is incubatedat 37° C. until MAP growth is detected.

The instant method is said to enhance MAP growth in that supplementationof a growth medium with a nicotine analog provides at least a 20%, 30%,40%, 50%, 60%, 70%, or 80% increase in growth as compared to a control,e.g., MAP not growth in the presence of a nicotine analog. As describedherein, growth enhancement can be determined and expressed as cumulativeGrowth Index.

As used in the context of the present invention, a “nicotine analog” isintended to mean a compound having the structure of Formula I.

wherein R₁ is NH₂ or O; and R₂ is absent, or a ribose group ornucleotide group. Nucleotide groups encompassed within the scope of theinvention include, but are not limited to, adenine dinucleotide ormononucleotide groups.

Exemplary nicotine analogs embraced by the invention include:

Nicotine analogs can be used at any appropriate amount, which may bedependent upon the analog selected. Particularly desirable amounts ofnicotine analogs used in the culture of MAP are in the range of 50 ng/mLto 100 μg/mL. The selection of an appropriate amount can be based uponthe data presented herein or by additional dose-response analyses.

As indicated, once MAP has been cultured under the conditions describedherein, it can be used for various purposes including research,diagnosis and the identification of anti-MAP agents for use in theprevention and/or treatment of a MAP infection. In this respect, theinstant invention further include a determination of antibioticsusceptibility to effectively select a treatment regime for a subjectfrom which a sample originated. To determine antibiotic susceptibility,the in vitro cultured MAP is contacted with a plurality of antibiotics(e.g., 2, 3, 4, 5, 10, or more). Antibiotic susceptibility testing canbe carried out using, e.g., the MIGT or BACTEC systems (bothBecton-Dickerson), or ESP II (Trek Diagnostic Systems, Cleveland, Ohio).While the BACTEC system is not highly automated and uses aradionucleotide (¹⁴C), this system is sensitive as it uses a medium thatis not very rich and therefore the antibiotics are not idiosyncraticallyabsorbed onto the proteins in the medium.

Having demonstrated that nicotine analogs enhance the growth of MAP,another feature of the present invention is a growth medium forenhancing the growth of MAP. The medium of the present inventionincludes a base growth medium in admixture with one or more nicotineanalogs described herein. For the purposes of the present invention, abase growth medium can be any medium known in the art to support thegrowth of MAP. Base growth media suitable for use in accordance with theinvention include, but are not limited to, BACTEC 460TB or MycobacteriaGrowth Indicator Tube (MIGT) media (Becton, Dickinson and Company,Cockeysville, Md.) as well as Middlebrook 7H9 broth enriched with oleicacid albumin dextrose complex, 0.4% casamino acids containing vitamins,40 μg/ml tryptophan, 0.5 μg/ml, mycobactin J, and 0.05% TWEEN 80. See,e.g., Harrisa, et al. (2006) FEMS Microbiol. Lett. 175:21-26.

For use in the enhancement of MAP growth, particular embodiments featurethe growth medium of the invention in the form of a kit. A kit accordingto the invention generally includes a base growth medium and one or morenicotine analogs described herein. The base growth medium and one ormore nicotine analogs can be provided in separate containers or in asingle container. The kit can further include other vitamins, proteins,minerals, sugars, or additives that support the growth of MAP. Inaddition, the kit can include instructions for mixing, sterilizing,and/or using the components of the kit. The kit can further includereagents, e.g., antibodies or primers, for detecting the presence ofMAP.

The invention is described in greater detail by the followingnon-limiting examples.

Example 1 Materials and Methods

In this study eight strains of mycobacteria were used, four of whichwere MAP. Two MAP strains had been isolated from humans with Crohn'sdisease. Dominic (ATCC 43545; Chiodini, et al. (1986) supra) and UCF 4(Naser, et al. (2004) supra). The other two MAP strains were fromruminants with Johne's disease ATCC 19698 (ATCC Rockville, Md.) and 303.The M. avium subspecies avium strains (hereinafter called M. avium) wereATCC 25291 (veterinary source) and M. avium 101 (Bertram, et al. (1986)J. Infect. Dis. 154:194-195). To study the M. tuberculosis complex, twoBioSafety level 2 strains, Bacillus Callmette Guerin (BCG) M. bovisKarlson & Lessel (ATCC 19015) and an avirulent M. tb strain; ATCC 25177were used.

Because it renders clinically resistant strains of MAP inappropriatelysusceptible to antimicrobials in cell culture (Damato & Collins (1990)Vet. Microbiol. 22:31-42), or interferes with growth and susceptibility(Greenstein, et al. (2009) supra), the detergent TWEEN 80 (recommendedto prevent mycobacterial clumping) was not used in cultures herein.

Cultures were processed according to conventional methods (Greenstein,et al. (2007) supra; Greenstein, et al. (2009) supra; Greenstein, et al.(2007) supra; Greenstein, et al. (2008) supra; Rastogi, et al. (1992)Antimicrob. Agents Chemother. 36:2843-2846; Greenstein, et al. (2009)Gut Pathogens 1:4). In brief, the radiometric ¹⁴CO₂ BACTEC® 460 system(Becton-Dickinson, Franklin Lakes, N.J.) was used. Each vial had theidentical concentration of all constituents, except for the amount oftest agent. Vials were assayed on a daily basis, quantifying the amountof ¹⁴C released as ¹⁴CO₂, by the integral detector in the BACTEC 460.The data were obtained as a manufacturer determined, arbitrary GrowthUnits (GU) of 0-999.

In this study, agents were dissolved in either Middlebrook 7H9 broth(DIFCO®,: Sparks, Md.) or 18 MΩ distilled water. Chemicals weredissolved, aliquoted, stored at −80° C., thawed, used once anddiscarded. Each experiment was performed with agents that had beendissolved in only water (all nicotine related molecules) or 7H9 broth(salicylic acid & PAS.)

As experimental controls, two well-established antibiotics were used toinhibit mycobacterial growth. Isoniazid (INH; Iso Nicotino Hydrazide),used to treat tuberculosis (Noufflard & Deslandes (1952) Ann. Inst.Pasteur. (Paris) 83:769-773) and leprosy (Lowe (1952) Lancet2:1012-1013) and para-amino salicylic acid (PAS) used to treattuberculosis Lehmann (1946) Lancet 1:15-16). In 1940 Bernheim firstreported the enhancement of growth of mycobacteria by salicylate(Bernheim (1940) Science 92:204). Accordingly, the experimental controlfor enhancement of growth was salicylic acid. Racemic (±) nicotine andit two enantiomers (+) and (−), were also analyzed, as were nicotinicacid, nicotinamide (Vitamin B3, an amide of nicotinic acid), and α- andβ-nicotinamide adenine dinucleotide (α- and β-NAD.) (All Sigma, StLouis. MO). Agents were studied at concentrations ranging from 0.1 to 64μg/ml.

For clarity and ease of understanding, data are presented in two ways;as the cumulative Growth Index (cGI) for an individual mycobacteriumfrom a single experiment (see Figures) and, alternatively, for a singlechemical the same data from all experiments were recalculated andpresented as the “percent change from control cGI” (see Tables). Anincrease was presented as “%+ΔcGI” and a decrease as “%−ΔcGI”. See,Greenstein, et al. (2007) supra for calculation.

Example 2 Use of Nicotine Analogs to Enhance Growth of MAP

The initial experimental control was salicylic acid, which showed adose-dependant enhancement of growth for M. avium ATCC 25291 (104%+ΔcGIat 64 μg/ml) and M. bovis (BCG) (95%+ΔcGI at 64 μg/ml). In contrast,only one of four MAP strains showed dose-dependant enhancement; MAP: 303(66%+ΔcGI at 64 μg/ml).

The inhibitory control was PAS (Lehmann (1946) supra), which exhibitedbactericidal activity against M. avium and BCG (≧94%−ΔcGI at 4 (four)μg/ml). PAS exhibited dose-dependent inhibition on MAP 19698 and MAP 303(80%−ΔcGI at 64 μg/ml).

The antibiotic inhibitory control for the structural analogs of nicotinewas INH (Robitzek & Selikoff (1952) Am. Rev. Tuberc. 65:402-428). INHwas bactericidal on M. tb (97%−ΔcGI at 1 (one) μg/ml) and haddose-dependent inhibition on BCG and M. avium: 101(≧97%−ΔcGI at 64μg/ml) (Table 1) In contrast, at the doses tested, INH has nodose-dependent inhibition on any MAP strain, although inhibitionoccurred by 64 μg/ml against all four MAP strains (Table 1, FIGS. 1 and2)

TABLE 1 M. tb MAP M. avium complex μg/ml Dominic* UCF-4 19698 303 25291101 BCG Tb 1 18%, 11% −5% −20% −18% 176% −48% −62% −97% 4  9%, 29%  2%−14%  −6%  89% −96% −25% −98% 16 −4%, 9%   −4% −17% −12% 189% −95% −63%−98% 64 −65%, −52% −35%  −63% −52% −90% −97% −99% −98% *Results from twoexperiments. Data are presented as percent increase (% ΔcGI) or decrease(% −ΔcGI) in cumulative Growth Index (cGI) from concomitant control. INHwas dissolved in water.

Nicotine, whether racemic (±) or either of its enantiomers (+) or (−),had neither enhancement nor an inhibitory effect on any of the eightmycobacterial strains, when studied at 1-64 μg/ml (Table 2; FIGS. 1-3).

TABLE 2 M. tb MAP M. avium complex μg/ml Dominic* UCF-4 19698 303 25291101 BCG Tb 1 −2%, 0%   −10% −20% 7% 106% 55% −65%  6% 4 29%, 30% −10%−20% 8%  63%  0% −41% −14% 16 1%, 6%  −6% −21% −22%  143% 15% −39% −10%64 −12%, 7%     9% −11% 7% 112%  5% −51%  2% *Results from twoexperiments. Data are presented as percent increase (% ΔcGI) or decrease(% −ΔcGI) in cumulative Growth Index (cGI) from concomitant control.Nicotine was purchased in aqueous solution.

Nicotinic acid is a component of tobacco (Griffith, et al. (1960)J.Biol. Chem. 235:3536-3538; Weidel (1873) Justus Liebig's Annalen derChemie and Pharmacie 165:328-349). Of the four non-MAP strains studied,nicotinic acid enhanced growth of M. avium, but not of the M.tuberculosis complex (Table 3, FIG. 3). Growth of MAP isolated fromhumans was enhanced by nicotinic acid (Dominic 225%+ΔcGI; UCF-4,92%+ΔcGI, at 64 μg/ml; Table 3, FIGS. 1 and 2). MAP from bovine sourcesshowed far less nicotinic acid induced enhancement than was observedwith the human MAP isolates (Table 3).

TABLE 3 M. tb MAP M. avium complex μg/ml Dominic* UCF-4 19698 303 25291101 BCG Tb 1 46%, 71% 48% −17%  −4%  98% 41% −45% −1% 4 133%, 181% 63%−4%  2% 170% 47% −61% 29% 16 195%, 198% 101%  12% −4% 167% 40% −45%  9%64 197%, 225% 92% 30% 22% 175% 56% −14% 15% *Results from twoexperiments. Data are presented as percent increase (% ΔcGI) or decrease(% −ΔcGI) in cumulative Growth Index (cGI) from concomitant control.Nicotinic acid was dissolved in water.

Initially, nicotinamide was studied at the same concentration as theother agents. However, against Dominic, the effect was high (≧100%+ΔcGIfor 1, 4, 16 and 64 μg/ml) and constant (Table 4, results from firstexperiment). Accordingly, 10-fold lower nicotinamide concentrations wereused for the remaining experiments (Table 4, second Dominic experimentand remaining seven columns, FIGS. 2 and 3).

TABLE 4 M. tb MAP M. avium complex μg/ml Dominic UCF-4 19698 303 25291101 BCG Tb 0.1 (1) 110%, 32% 20% −6% −5%  23% 6% −71% −1% 0.4 (4) 115%,98% 52% −2% −28%  105% −21%  −19% 11% 1.6 (16) 125%, 156% 73% −8% 11%157% 7% −43%  4% 6.4 (64) 106%, 156% 79% −7%  3% 144% 58%  −42% 10% Dataare presented as percent increase (% ΔcGI) or decrease (% −ΔcGI) incumulative Growth Index (cGI) from concomitant control. Nicotinamide wasdissolved in water. Note that, uniquely for this study, the nicotinamidewas studied at a 10-fold greater dilution than the other agents studied.The single exception is the index Dominic experiment (second data set),wherein the standard dilutions (shown in parentheses) were used.

For M. avium (25291 & 101), nicotinamide caused enhancement of growth(Table 4). In contrast, nicotinamide had no effect on the M.tuberculosis complex (BCG & M. tb) (Table 4, FIG. 3). Comparable toobservations with nicotinic acid (Table 3), nicotinamide enhanced MAPisolated from humans, but had no effect on MAP isolated from ruminants(Table 4, FIG. 2).

Two molecules that contain nicotinamide as an integral component oftheir structure were also analyzed, α- and β-NAD. The human MAP isolates(Dominic & UCF-4), whose growth was enhanced by nicotinamide itself,were likewise enhanced by α-NAD (Dominic, 135%+ΔcGI; UCF-4, 81%+ΔcGI, at64 μg/ml) and β-NAD (Dominic, 150%+ΔcGI; UCF-4, 79%+ΔcGI, at 64μg/ml)(Tables 5 and 6, FIGS. 1 and 2). In contrast, α- and β-NAD hadlittle or no effect on the other six strains studied, including the twoMAP bovine isolates (Tables 5 and 6, FIG. 3).

TABLE 5 M. tb MAP M. avium complex μg/ml Dominic* UCF-4 19698 303 25291101 BCG Tb 1   25%, −16% 18% −16% 8%  −2%  39% −51%  2% 4 79%, 35% 35% −6% 9% 160% −15% −60% −6% 16 104%, 78%  65% −17% −8%  131% −21% −50%−1% 64 135%, 115% 81%  13% −18%  147% −14% −48%  0% *Results from twoexperiments. Data are presented as percent increase (% ΔcGI) or decrease(% −ΔcGI) in cumulative Growth Index (cGI) from concomitant control.α-NAD was dissolved in water.

TABLE 6 M. tb MAP M. avium complex μg/ml Dominic* UCF-4 19698 303 25291101 BCG Tb 1 13%, 24% 59% 5% 18% 123%  18% −72% 20% 4 60%, 87% 79% 23% 34% 66% 24%  1% 21% 16 130%, 122% 95% 6% 18% −37%  27% −47%  9% 64 114%,150% 79% 8%  1% 42% 22% −59% 14% *Results from two experiments. Data arepresented as percent increase (% ΔcGI) or decrease (% −ΔcGI) incumulative Growth Index (cGI) from concomitant control. β-NAD wasdissolved in water.

In IBD, tobacco use results in paradoxical responses amongst patientswho have CD or UC. These effects have no accepted mechanism of action.Because of different delivery methods and use patterns, the bioavailabledose of tobacco products that will occur in the human host will varyconsiderably (Pavia, et al. (2000) J. Med. Microbiol. 49:675-676). Inthis study, it was determined whether some of the ≧4000 molecules foundin tobacco could perturb MAP growth, thereby providing a rationalexplanation for some tobacco related IBD effects.

Nicotine is the most commonly studied molecule associated with tobacco.Prior in vitro results are discordant. Enhanced growth of M.tuberculosis, M. Kansasii, M. scrofulaceum, M. avium and M.intracellulare, occurs at extremely high doses of nicotine (5 mg/ml)(Kotian, et al. (1984) Ind. J. Tuberc. 31:151-158). In contrast,inhibition of bacteria (including M. phlei) and fungal growth isobserved at lower doses of nicotine (100-250 μg/ml) (Zelitch (1955) J.Biol. Chem. 216:553-575). Bactericidal inhibition of a single MAP strainwas reported by 2 μg/ml nicotine (Naser, et al. (2001) supra), using thesame BACTEC 460® system herein; however with supplementation of OADC(oleic acid, bovine serum albumin, dextrose and catalase). In thisstudy, no effect on growth was observed for nicotine at 1-64 μg/ml oneight strains of three mycobacterial species.

Nicotinic acid is a naturally occurring constituent of tobacco(Griffith, et al. (1960) supra). The nicotinic acid dose-dependentenhancement observed herein occurred in all four MAP strains. However,the effect was most pronounced in the MAP strains isolated from humans.Additionally, nicotinic acid enhanced one M. avium strain, (25291).Nicotinamide is the amide of nicotinic acid. Both human MAP isolateswere remarkably susceptible to nicotinamide growth enhancement. Incontrast, neither of the bovine MAP isolates were enhanced at the doseof nicotinamide studied. Likewise, although M. avium growth wasenhanced, nicotinamide had no effect on the representatives of the M.tb. complex. It was concluded that the enhanced response of mycobacteriato nicotinic acid and nicotinamide was both species-, as well asstrain-, dependent.

Intact α-NAD enhanced MAP isolated from humans as well as M. avium25291. β-NAD enhanced only the two human MAP isolates. Tobacco isparticularly rich in the enzyme glycolic acid reductase (Zelitch (1955)supra). This enzyme reduces diphosphopyridine nucleotides to theirconstituent components (Zelitch (1953) J. Biol. Chem. 201:719-726;Zelitch & Ochoa (1953) J. Biol. Chem. 201:707-718). The glycolic acidreductase fraction of tobacco may, in vivo, cleave α- and (β-NAD torelease nicotinamide. This could provide an additional, indirect,mechanism whereby tobacco products enhance the growth of MAP.Parenthetically, catalase markedly enhances the effect of glycolic acidreductase (Zelitch & Ochoa (1953) supra). This observation may, in part,account for the differences observed between the effect of nicotine inthe present study and a prior study where catalase was added to theculture medium (Naser, et al. (2001) supra).

Overall, the analysis herein indicates that the use of tobacco, or itsindividual components, are compatible with either enhancement orinhibition of the growth of mycobacteria both in vitro and in vivo.

1. A method for enhancing the growth of Mycobacterium avium subspeciesparatuberculosis (MAP) comprising culturing MAP in the presence of anicotine analog thereby enhancing the growth of MAP.
 2. The method ofclaim 1, wherein the nicotine analog has the structure

wherein R₁ is NH₂ or O, and R₂ is absent, or a ribose or nucleotidegroup.
 3. The method of claim 2, wherein the nicotine analog isnicotinic acid, nicotinamide, α-nicotinamide adenine dinucleotide, orβ-nicotinamide adenine dinucleotide.
 4. The method of claim 1, whereinthe MAP is from a biological sample.
 5. A growth medium for enhancingthe growth of Mycobacterium avium subspecies paratuberculosis (MAP)comprising a base growth medium the supports the growth of MAP inadmixture with at least one nicotine analog.
 6. The growth medium ofclaim 5, wherein the nicotine analog has the structure

wherein R₁ is NH₂ or O, and R₂ is absent, or a ribose or nucleotidegroup.
 7. The growth medium of claim 6, wherein the nicotine analog isnicotinic acid, nicotinamide, α-nicotinamide adenine dinucleotide, orβ-nicotinamide adenine dinucleotide.
 8. A kit comprising a base growthmedium and at least one nicotine analog.
 9. The kit of claim 8, whereinthe nicotine analog has the structure

wherein R₁ is NH₂ or O, and R₂ is absent, or a ribose or nucleotidegroup.
 10. The kit of claim 9, wherein the nicotine analog is nicotinicacid, nicotinamide, α-nicotinamide adenine dinucleotide, orβ-nicotinamide adenine dinucleotide.